Rice glutelin gene promoters

ABSTRACT

A nucleic acid containing a glutelin gene promoter. Disclosed are transformed plant cells and transgenic plants containing a nucleic acid that includes the promoter operably linked to a sequence encoding heterologous protein. Also disclosed are methods of making the transformed plant cells and transgenic plants and methods for expressing a polypeptide.

RELATED APPLICATION

This application claims priority to U.S. application Ser. No.60/460,037, filed Apr. 3, 2003, the contents of which are incorporatedherein by reference.

BACKGROUND

Transgenic plants and transformed plant cells have been used forproducing recombinant proteins, such as enzymes, antibodies, vaccines,and therapeutic proteins. See, e.g., Fischer et al., 2000, TransgenicRes. 9:279-299; Giddings et al., 2000, Nat. Biotechnol. 18:1151-1155;Stoger et al., 2002, Curr. Opin. Biotechnol. 13:161-166; and Ma et al.,2003, Nat. Rev. Genet. 4:794-805. The plant-based expression systemsoffer several advantages over others. For example, they (1) have verylittle risk of contamination with mammalian pathogens or bacterialtoxins, (2) are capable of protein post-translation modification, (3)are more economic than bioreactor-based systems, (4) can be scaled up atrelatively low costs, (5) can be developed within a short timeframe, and(6) require no or partial purification if the recombinant proteins areused directly as foods, feed supplements, or raw materials for use. Inparticular, since plant cells are able to properly process recombinantmammalian proteins, these systems have been used to produce functionaland complex mammalian proteins (Sijmons et al., 1990, Biotechnology8:217-221; Hiatt et al., Nature 342:76-78 and During et al., Plant Mol.Biol. 15:281-293). Nonetheless, they also have limitations. In manycases, the amount of expressed protein ranges from 0.001% to 0.1% oftotal soluble proteins (Daniell et al., 2001, Trends Plant Sci.6:219-226), which is too low for commercial production. Thus, there is aneed for a high yield plant expression system.

SUMMARY

This invention is based on the discovery that novel rice glutelin B genepromoters (GluB-1 promoters) drive high level expression of aheterologous recombinant protein in a plant cell. An exemplary GluB-1promoter (SEQ ID NO: 2) is shown below. It corresponds to nucleotides(nt) −1307 to +36 of the rice glutelin B gene (GluB), where position +1represents the transcription start site of the gene. −1307 gatctcgatttttgaggaat tttagaagtt gaacagagtc aatcgaacag acagttgaag (SEQ ID NO: 2)−1247 agatatggat tttctaagat taattgattc tctgtctaaa gaaaaaaagt attattgaat−1187 taaatggaaa aagaaaaagg aaaaagggga tggcttctgc tttttgggct gaaggcggcg−1127 tgtggccagc gtgctgcgtg cggacagcga gcgaacacac gacggagcag ctacgacgaa−1067 cgggggaccg agtggaccgg acgaggatgt ggcctaggac gagtgcacaa ggctagtgga−1007 ctcggtcccc gcgcggtatc ccgagtggtc cactgtctgc aaacacgatt cacatagagc−947 gggcagacgc gggagccgtc ctaggtgcac cggaagcaaa tccgtcgcct gggtggattt−887 gagtgacacg gcccacgtgt agcctcacag ctctccgtgg tcagatgtgt aaaattatca−827 taatatgtgt ttttcaaata gttaaataat atatataggc aagttatatg ggtcaataag−767 cagtaaaaag gcttatgaca tggtaaaatt acttacacca atatgcctta ctgtctgata−707 tattttacat gacaacaaag ttacaagtac gtcatttaaa aatacaagtt acttatcaat−647 tgtagtgtat caagtaaatg acaacaaacc tacaaatttg ctattttgaa ggaacactta−587 aaaaaatcaa taggcaagtt atatagtcaa taaactgcaa gaaggcttat gacatggaaa−527 aattacatac accaatatgc tttattgtcc ggtatatttt acaagacaac aaagttataa−467 gtatgtcatt taaaaataca agttacttat caattgtcaa gtaaatgaaa acaaacctac−407 aaatttgtta ttttgaagga acacctaaat tatcaaatat agcttgctac gcaaaatgac−347 aacatgctta caagttatta tcatcttaaa gttagactca tcttctcaag cataagagct−287 ttatggtgca aaaacaaata taatgacaag gcaaagatac atacatatta agagtatgga−227 cagacatttc tttaacaaac tccatttgta ttactccaaa agcaccagaa gtttgtcatg−167 gctgagtcat gaaatgtata gttcaatctt gcaaagttgc ctttcctttt gtactgtgtt−107 ttaacactac aagccatata ttgtctgtac gtgcaacaaa ctatatcacc atgtatccca−47 agatgctttt ttattgctat ataaactagc ttggtctgtc tttgaactca catcaattag+14 cttaagtttc cataagcaag tac

Accordingly, the invention features a nucleic acid containing a GluB-1promoter. A GluB-1 promoter as used herein refers to two types ofnucleic acids.

The first type GluB-1 promoter is an isolated nucleic acid sequence that(1) contains SEQ ID NO: 1 (corresponding to nt −1307 to −1 of thesequence list above) or its complement, and (2) is 1,307 to 2,300 (i.e.,any integer between 1,307 and 2,300, inclusive) nucleotides in length.An “isolated nucleic acid” is a nucleic acid the structure of which isnot identical to that of any naturally occurring nucleic acid. The termtherefore covers, for example, (a) a DNA which has the sequence of partof a naturally occurring genomic DNA molecule but is not flanked by bothof the coding sequences that flank that part of the molecule in thegenome of the organism in which it naturally occurs; (b) a nucleic acidincorporated into a vector or into the genomic DNA of a prokaryote oreukaryote in a manner such that the resulting molecule is not identicalto any naturally occurring vector or genomic DNA; (c) a separatemolecule such as a cDNA, a genomic fragment, a fragment produced by PCRamplification, or a restriction fragment; and (d) a recombinantnucleotide sequence, e.g., a nucleotide sequence containing heterologoussequences or encoding a fusion protein.

The second type GluB-1 promoter is a nucleic acid sequence that (1)contains tatccatatcca (SEQ ID NO: 9) or cctacgtggc (SEQ ID NO: 14), orits complement, and (2) is at least 20 (i.e., any integer no less than20, e.g., 40, 60, 100, 500, 1,000, or 2,000) nucleotides in length. SEQID NOs: 9 and 14 are mutant forms of SEQ ID NO: 2 regions from nt −54 to−43 (SEQ ID NO: 8) and from nt −82 to −73 (SEQ ID NO: 13), respectively.Shown below is the nt −92 to −28 region of SEQ ID NO: 2. This region,i.e., SEQ ID NO: 3, spans SEQ ID NOs: 8 and 13 (both underlined). SEQ IDNO: 3              -82    -73              -57 -54      -43  -39           |          |               |  |          |   | -92atatattgtctgtacgtgca acaaactatatcaccatg tatcccaagatg cttttttattgctat-28:           SEQ ID NO: 13                 SEQ ID NO: 8

In one mutant, the region corresponding to nt −57 to −39 of SEQ ID NO: 3is atgTATCCATATCCActtt (SEQ ID NO: 10) or cctTATCCATATCCAcgcc (SEQ IDNO: 11). Both SEQ ID NOs: 10 and 11 include SEQ ID NO: 9 (shown in uppercase). Accordingly, a GluB-1 promoter of this invention can contain amutant form of SEQ ID NO: 3, which has one or more of SEQ ID NOs: 10,11, and 14. Examples of such a promoter include the following sequences:(SEQ ID NO: 4) GM1:-92atatattgtctgtacgtgcaacaaactatatcaccatgTATCCATATCCActtttttattgctat-28           SEQ ID NO: 13                 SEQ ID NO: 10 (SEQ ID NO: 5)GM2: -92atatattgtctgtacgtgcaacaaactatatcacc cctTATCCATATCCAcgcctttattgctat-28            SEQ ID NO: 13                 SEQ ID NO: 11(SEQ ID NO: 6) GM3: -92atatattgtc cctacgtggc acaaactatatcacccctTATCCATATCCAcgcc tttattgctat-28            SEQ ID NO:14                 SEQ ID NO: 11Also within the scope of this invention are mutant forms of SEQ ID NO: 1that contains SEQ ID NOs: 4-5 (i.e., SEQ ID NO: 15-17, respectively) andmutant forms of SEQ ID NO: 2 that contains SEQ ID NOs: 4-5 (i.e., SEQ IDNO: 18-20, respectively).

Each of the above-described nucleic acid sequences can be included in apromoter to drive the expression of a heterologous gene in a plant cellor transgenic plant. Thus, the invention features a vector containing apromoter that includes one of the above described nucleic acids. Thepromoter can be further operatively linked to a recombinant nucleic acidthat encodes a heterologous protein of interest.

The invention further features a transformed plant cell that contains(1) the above described promoter sequence and (2) a recombinant nucleicacid that encodes a heterologous protein and is operatively linked tothe promoter sequence. In one embodiment, the plant cell is a monocotplant cell, such as a cereal plant cell (e.g., a rice cell, a corn cell,a wheat cell, a barley cell, an oat cell, or a sorghum cell). In anotherembodiment, the plant cell is a dicot plant cell, e.g., a tobacco cell,a potato cell, a tomato cell, or a soybean cell. To make the plant celldescribed above, one can introduce into a plant cell the promotersequence and the recombinant nucleic acid. In one embodiment, thetransformed plant cell is a cultured cell. Exemplary cultured cellsinclude protoplasts, calli, suspension cells, and tissues.

The invention also features a transgenic plant whose genome contains (1)one of the above-described promoter sequences and (2) a recombinantnucleic acid that encodes a heterologous protein and is operativelylinked to the promoter sequence. The plant can be (1) a monocot plant,including a cereal plant, e.g., rice, corn, wheat, barley cell, oat, orsorghum; or (2) a dicot plant, e.g., tobacco, potato, tomato, orsoybean. To make such a transgenic plant, one can introduce into a plantcell the promoter sequence and the recombinant nucleic acid, andcultivate the cell to generate a plant.

Finally, the invention features a method of expressing a polypeptide ina cell. The method includes (1) introducing into a host cell arecombinant nucleic acid encoding a polypeptide, wherein the recombinantnucleic acid is operatively linked to a GluB-1 promoter sequencedescribed above, (2) culturing the host cell under conditions permittingexpression of the polypeptide, and (3) recovering the polypeptide. Thehost cell can be cultured in the presence or absence of sugar toregulate the expression level of the polypeptide.

The details of one or more embodiments of the invention are set forth inthe accompanying description below. Other advantages, features, andobjects of the invention will be apparent from the detailed descriptionand the claims.

DETAILED DESCRIPTION

The present invention relates to native GluB-1 promoter sequences, aswell as their variant sequences. These sequences and variants can beused in generating plant cells or transgenic plants for producingrecombinant protein.

Accordingly, the invention includes a transformed plant cell ortransgenic plant containing a recombinant nucleic acid that encodes aheterologous protein. Expression of the protein is under the control ofone of theGluB-1 promoter sequences described above. The plant cell canbe a dicot plant cell or a monocot plant cell.

A transformed plant cell of the invention can be produced by introducinginto a plant cell a vector containing a GluB-1 promoter sequence that isoperatively linked to a recombinant nucleic acid encoding a desiredheterologous protein. Techniques for transforming a wide variety ofplant cells are well known in the art and described in the technical andscientific literature. See, for example, Weising et al., 1988, Ann. Rev.Genet. 22:421-477. To express a heterologous protein in a plant cell,the gene can be linked to a GluB-1 promoter sequence, as well as othertranscriptional and translational initiation regulatory sequences thatwill direct the transcription of the gene and translation of the encodedprotein in the plant cell.

For example, for over-expression, a constitutive plant promoter may beemployed in addition to a GluB-1 promoter. A “constitutive” promoter isactive under most environmental conditions and states of celldifferentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumafaciens, theACT 11 and Cat3 promoters from Arabidopsis (Huang et al., 1996, PlantMol. Biol. 33:125-139 and Zhong et al., 1996, Mol. Gen. Genet.251:196-203), the stearoyl-acyl carrier protein desaturase gene promoterfrom Brassica napus (Solocombe et al., 1994, Plant Physiol.104:1167-1176), the GPc1 and Gpc2 promoters from maize (Martinez et al.,1989, J. Mol. Biol. 208:551-565 and Manjunath et al., 1997, Plant Mol.Biol. 33:97-112).

Alternatively, a plant promoter may be employed to direct expression ofthe heterologous gene in a specific cell type (i.e., tissue-specificpromoters) or under more precise environmental or developmental control(i.e., inducible promoters). Examples of environmental conditions thatmay affect transcription by inducible promoters include anaerobicconditions, elevated temperature, the presence of light, spray withchemicals or hormones, or infection of a pathogen. Examples of suchpromoters include the root-specific ANR1 promoter (Zhang and Forde,1998, Science 279:407) and the photosynthetic organ-specific RBCSpromoter (Khoudi et al., 1997, Gene 197:343).

For proper polypeptide expression, a polyadenylation region at the3′-end of the coding region should be included. The polyadenylationregion can be derived from the natural gene, from a variety of otherplant genes, or from T-DNA.

A marker gene can also be included to confer a selectable phenotype onplant cells. For example, the marker gene may encode a protein thatconfers biocide resistance, antibiotic resistance (e.g., resistance tokanamycin, G418, bleomycin, hygromycin), or herbicide resistance (e.g.,resistance to chlorosulfuron or Basta).

A recombinant nucleic acid that encodes a heterologous protein may beintroduced into the genome of a desired plant host cell by a variety ofconventional techniques. For example, the recombinant nucleic acid maybe introduced directly into the genomic DNA of a plant cell usingtechniques such as protoplast electroporation and microinjectionl, orthe recombinant nucleic acid can be introduced directly to plant tissueusing ballistic methods, such as DNA particle bombardment. A transformedplant cell of the invention can be produced by introducing into a plantcell a vector containing a GluB-1 promoter sequence that is operativelylinked to a recombinant nucleic acid encoding a desired heterologousprotein. Microinjection techniques are well documented in the scientificand patent literature. Introduction of a recombinant nucleic acid usingpolyethylene glycol precipitation is described in Paszkowski et al.,1984, EMBO J. 3:2717-2722. Electroporation techniques are described inFromm et al., 1985, Proc. Natl. Acad. Sci. USA 82:5824. Ballistictransformation techniques are described in Klein et al., 1987, Nature327:70-73.

Alternatively, the recombinant nucleic acid may be combined withsuitable T-DNA flanking regions and introduced into a conventionalAgrobacterium tumefaciens host vector. The virulence functions of theAgrobacterium tumefaciens host will direct the insertion of theheterologous gene and adjacent marker into the plant cell DNA when thecell is infected by the bacteria. Agrobacterium tumefaciens-mediatedtransformation techniques, including disarming and use of binaryvectors, are well known in the art. See, e.g., Horsch et al., 1984,Science 233:496-498, Fraley et al., 1983, Proc. Natl. Acad. Sci. USA80:4803, and Gene Transfer to Plants, Potrykus, ed., Springer-Verlag,Berlin, 1995.

The presence and copy number of the gene encoding a desired heterologousprotein in a transgenic plant can be determined using methods well knownin the art, e.g., Southern blotting analysis. Expression of theheterologous gene in a transgenic plant may be confirmed by detectingthe corresponding heterologous mRNA or protein in the transgenic plantby methods well known in the art.

Transformed plant cells that are prepared by any of the above-describedtransformation techniques can be cultured to regenerate a whole plant.Such regeneration techniques rely on manipulation of certainphytohormones in a tissue culture growth medium, typically relying on abiocide or herbicide marker that has been introduced together with theheterologous gene. Plant regeneration from cultured protoplasts isdescribed in Evans et al., Protoplasts Isolation and Culture, Handbookof Plant Cell Culture, pp. 124-176, MacMillilan Publishing Company, NewYork, 1983; and Binding, Regeneration of Plants, Plant Protoplasts, pp.21-73, CRC Press, Boca Raton, 1985. Regeneration can also be obtainedfrom plant callus, explants, organs, or parts thereof. Such regenerationtechniques are described generally in Klee et al., 1987, Ann. Rev. PlantPhys. 38:467-486. Once the heterologous gene has been confirmed to bestably incorporated in the genome of a transgenic plant, it can beintroduced into other plants by sexual crossing. One or more standardbreeding techniques can be used, depending upon the species to becrossed.

The above-described plant cell/plant can be either dicot or monocot.There are advantages for using a cereal plant as a host for recombinantproteins. Cereal crops (e.g., rice, corn and wheat) are grown worldwide.Further, there are well-established agricultural practices for theirproduction, distribution and processing. They can be transported anddistributed over long distances at ambient temperatures and have a longshelf life (Stoger et al., 2002, Curr Opin Biotechnol 13:161-166; andStreatfield et al., Vaccine 19:2742-2748). They have high annual yieldsand are therefore suitable for producing a large amount of protein.Unlike green tissues (e.g., tobacco and alfalfa), they have (1) lowerlevels of toxic secondary metabolites and (2) lower hydrolytic profilesand lower level of proteins and lipids, thereby facilitating proteinpurification and enhancing protein stability, respectively (Delaney,2002, In: Plants as Factories for Protein Production Hood, E. E. andHoward, J. A., eds, Netherlands: Kluwer Academic, pp. 139-158).Recombinant proteins produced in cereals are stable during storage, dueto very low water contents. Rice is one of the most important crops andstaple foods in the world. As a protein expression system, rice hasseveral advantages over other cereal crops (e.g., wheat, barley andrye), including efficient transformation technology, availability of avariety of constitutive and regulated promoters, greater biomass (yieldper unit area), and lower production costs (Stoger et al., 2002, Curr.Opin. Biotechnol. 13:161-166). Additionally, since rice is aself-pollinated crop, gene flow between wild type and transgenic speciesthrough cross-pollination is much limited. In other words, geneticcontamination is minimized.

When using rice seeds as a platform for producing recombinant proteins,it is important to use promoters that control gene expression indeveloping rice seeds. Glutelin is the most abundant seed storageprotein in rice, consisting 60%-80% of total endosperm proteins inmature seeds (Wen et al., 1985, Plant Physiol 78:172-177; and Li et al.,1993, Plant Cell Physiol. 34:385-390). The rice glutelin is encoded by afamily of approximately 10 genes per haploid rice genome (Okita et al.,1989, J. Biol. Chem. 264: 12573-12581; and Takaiwa et al., 1991, Jpn. J.Genet. 66:161-171). Based on the sequence similarity, rice glutelingenes could be categorized into two subfamilies, designated as GluA andGluB. Each of the GluA and GluB subfamilies contains at least fourgenes, with coding regions sharing 75%-95% homology. Several glutelingene promoters have been demonstrated to be specifically active indeveloping rice endosperms (Wu et al., 2000, Plant J 23:415-421; Wu etal., 1998, Plant Cell Physiol 39: 885-889; and Wu et al., 1998, Plant J14:673-683). The GluB promoter possesses higher activity than the GluApromoter and its cis-acting elements have been analyzed. A 245-bp regionin the GluB promoter is necessary for conferring endosperm-specificexpression in developing rice seeds (Yoshihara et al., 1996, Plant CellPhysiol 37: 107-111; Yoshihara et al., 1996, FEBS Lett. 383:213-218).This region contains two AACA motifs (AACAAAC), one GCN4 motif(TGAGTCA), and one G-box. The GCN4 motif is a determinant forendosperm-specific expression, while the AACA motif and G box areresponsible for quantitative regulation of the promoter.

Expressing proteins in rice seeds requires a strong, developing riceseed-specific promoter. Although several glutelin promoters have beenused for expressing recombinant proteins in transgenic rice and barleyseeds, the yields of these proteins are generally too low (ranging fromless than 0.1% to 1% of total soluble protein) for practical use (Gotoet al., 1999, Nat. Biotechnol. 17:282-286).

Various strategies can be employed to increase promoter activity. One ofthem is to modify cis-acting elements in the promoter. It has been shownthat the sugar response sequence (SRS) in the promoter of a riceα-amylase gene, αAmy3, functions as a transcriptional enhancer for bothhomologous and heterologous promoters in transient expression and stabletransformation assays (Lu et al., 1998, J. Biol. Chem. 273:10120-10131;and Chen et al., 2002, J. Biol. Chem. 277:13641-13649). According toU.S. Pat. No. 5,460,952, rice α-amylase gene promoters aresugar-down-regulated and can be used to express recombinant protein inplant cells after the cells have been subjected to sugar starvation.

The sugar starvation-inducible expression system can be used forproduction of regulatory proteins. When production of recombinantprotein in large quantity becomes detrimental to cell growth, thissystem is particularly advantageous. Cells can be grown in a regularmedium containing sucrose, and little or no recombinant protein isproduced. The cells can then be subjected to sucrose starvation for ashort period of time to induce expression of recombinant proteins, andthe growth can be resumed by switching into a sucrose-containing medium.On the hand, it is desired to produce proteins from cells cultured inthe presence sucrose since it is tedious to switch betweensucrose-containing and sucrose-lacking culture medium. The inventionprovides such an expression system that can be up-regulated by sugar,e.g., sucrose.

As indicated in the examples below, both the rice α-amylase genepromoters and glutelin gene promoters lead to high level expression ofrecombinant protein (up to 12% of total proteins) in media lacking andcontaining sucrose, respectively. Note that these two types of promotershave similar levels of efficiency. If the overexpression of the proteinaffects cell growth, the α-amylase gene promoter can be used. Otherwise,both types of promoters can be used. Cell growth and protein yield canthen be compared and evaluated to select a more efficient andappropriate system.

The specific examples below are to be construed as merely illustrative,and not limitative of the remainder of the disclosure in any waywhatsoever. Without further elaboration, it is believed that one skilledin the art can, based on the description herein, utilize the presentinvention to its fullest extent. All publications cited herein arehereby incorporated by reference in their entirety.

EXAMPLE 1

A GluB-1 promoter containing SEQ ID NO: 1 was used to generatetransformed rice cells and transgenic rice.

Plant Material

The rice variety used in this example was Oryza sativa L. cv. Tainung67. Immature seeds were dehulled, sterilized with 2.4% NaOCl for 1 hour,washed extensively with sterile water, and placed on an N6D agar mediumfor callus induction according to the method described in Toki, 1997,Plant Mol. Biol. Rep. 15, 16-21. After one month, callus derived fromscutella were subcultured in a fresh N6D medium for transformation, orin a liquid MS medium (Murashige et al., 1962, Physiol. Plant15:473-497) containing 3% sucrose and 10 μM 2,4-D for establishing asuspension cell culture in the manner described in Yu et al., 1991, J.Biol. Chem. 266:21131-21137.

Plasmid construction

A 1351-bp glutelin gene promoter (shown below, nt 1-1351) wasPCR-amplified using rice genomic DNA as a template. The forward primerand reverse primer were B 1-5 (5′-GGGGAATTCGATCTCGATTTTTGAGGAAT-3′,EcoRI site underlined) and B1-sp (5′GGGGGATCCGGGATTAAATAGCTGGGCCA-3′,BamHI site underlined), respectively. A 75-bp sequence encoding aputative signal peptide (sp) sequence is also shown below (nt1352-1426). The putative 25-amino acid signal peptide cleavage site waspredicted based on a statistical method (von Heijne, 1985, J. Mol. Biol.184, 99-105). 1 GATCTCGATT TTTGAGGAAT TTTAGAAGTT GAACAGAGTC AATCGAACAGACAGTTGAAG 61 AGATATGGAT TTTCTAAGAT TAATTGATTC TCTGTCTAAA GAAAAAAAGTATTATTGAAT 121 TAAATGGAAA AAGAAAAAGG AAAAAGGGGA TGGCTTCTGC TTTTTGGGCTGAAGGCGGCG 181 TGTGGCCAGC GTGCTGCGTG CGGACAGCGA GCGAACACAC GACGGAGCAGCTACGACGAA 241 CGGGGGACCG AGTGGACCGG ACGAGGATGT GGCCTAGGAC GAGTGCACAAGGCTAGTGGA 301 CTCGGTCCCC GCGCGGTATC CCGAGTGGTC CACTGTCTGC AAACACGATTCACATAGAGC 361 GGGCAGAGGC GGGAGCCGTC CTAGGTGCAC CGGAAGCAAA TCCGTCGCCTGGGTGGATTT 421 GAGTGACACG GCCCACGTGT AGCCTCACAG CTCTCCGTGG TCAGATGTGTAAAATTATCA 481 TAATATGTGT TTTTCAAATA GTTAAATAAT ATATATAGGC AAGTTATATGGGTCAATAAG 541 CAGTAAAAAG GCTTATGACA TGGTAAAATT ACTTACACCA ATATGCCTTACTGTCTGATA 601 TATTTTACAT GACAACAAAG TTACAAGTAC GTCATTTAAA AATACAAGTTACTTATCAAT 661 TGTAGTGTAT CAAGTAAATG ACAACAAACC TACAAATTTG CTATTTTGAAGGAACACTTA 721 AAAAAATCAA TAGGCAAGTT ATATAGTCAA TAAACTGCAA GAAGGCTTATGACATGGAAA 781 AATTACATAC ACCAATATGC TTTATTGTCC GGTATATTTT ACAAGACAACAAAGTTATAA 841 GTATGTCATT TAAAAATACA AGTTACTTAT CAATTGTCAA GTAAATGAAAACAAACCTAC 901 AAATTTGTTA TTTTGAAGGA ACACCTAAAT TATCAAATAT AGCTTGGTACGCAAAATGAC 961 AACATGCTTA CAAGTTATTA TCATCTTAAA GTTAGAGTCA TCTTCTCAAGCATAAGAGCT 1021 TTATGGTGCA AAAACAAATA TAATGACAAG GCAAAGATAC ATACATATTAAGAGTATGGA 1081 CAGACATTTC TTTAACAAAC TCCATTTGTA TTACTCCAAA AGCACCAGAAGTTTGTCATG 1141 GCTGAGTCAT GAAATGTATA GTTCAATCTT GCAAAGTTGC CTTTCCTTTTGTACTGTGTT 1201 TTAACACTAC AAGCCATATA TTGTCTGTAC GTGCAACAAA CTATATCACCATGTATCCGA 1261 AGATGCTTTT TTATTGCTAT ATAAACTAGC TTGGTCTGTC TTTGAACTCACATCAATTAG 1321 CTTAAGTTTC CATAAGCAAG TACAAATAGC TATGGCGAGT TCCGTTTTCTCTCGGTTTTC 1381 TATATACTTT TGTGTTCTTC TATTATGCCA TGGTTCTATG GCCCAGCTATTTAATCCC

The PCR-amplified GluB-1 promoter-signal peptide sequence was digestedwith EcoRI and BamHI and subcloned into the corresponding sites inpBluescript (Strategene) to generate pBS-G and pBS-Gp. The promotersequence was then placed upstream of the coding region of Apu to maketranslational fusion constructs.

A sequence encoding a truncated Apu amino acid 75 to 1029 wasPCR-amplified using genomic DNA of T. ethanolicus 39E as a template(Mathupala et al., 1993, J. Biol. Chem. 268:16332-16344). The forwardprimer and reverse primer were 5′-CGGGATTCCTTAAGCTTGCATCTTGA-3′ (BamHIsite underlined) as and 5′-CCGGCGGCCGCCTACATATTTTCCCCTTGGCCA-3′ (NotIsite underlined), respectively. The PCR product was digested with BamHIand NotI and fused downstream of the GluB-1 promoter-signal peptidesequence in pBS-Gp to make translational fusion to generate pBS-Gp-Apu.

The nopaline synthase gene germinator (Nos 3′) was PCR-amplified usingpBI221 (Clontech) as the DNA template. The forward and reverse primerswere 5′-TCCGAGCTCCAGATCGTTCAAACATTT-3′ (SacI site underlined) and5′-AGCGAGCTCGATCGATCTAGTAACAT-3′ (SacI underlined), respectively. TheNos 3′UTR was digested with SacI and fused downstream of Apu inpBS-Gp-Apu to generate pBS-Gp-Apu-Nos.

A 1.2 kb αAmy8 promoter-signal peptide sequence was excised with SalIand HindIII from pAG8 (Chan et al., 1993, Plant Mol. Biol. 22:491-506.)and subcloned into pBluescript to generate pBS/8sp. The αAmy8 3′UTRswere PCR-amplified from a RAMYG6a plasmid (Yu et al., 1992, Gene122:247-253) using 5′-CGCCGCGGTAGCTTTAGCTATAGCGAT-3′ (SacII siteunderlined, forward primer) and 5′-TCCCCGCGGGTCCTCTAAGTGAACCGT-3′ (SacIIunderlined, reverse primer). RAMYG6a contains the 3′ half codingsequence and 3′ flanking region of αAmy8 genomic DNA and was generatedby the screening of a rice genomic DNA library (Clontech) using αAmy8-Cas a probe. The αAmy8 3′UTRs were subcloned into the SacII sites ofpBS/8sp to generate pBS/8sp8U. The truncated apu was cut with BamHI andNotI and subcloned into the same sites in pBS-8sp8U to generatepBS-αAmy8-sp-Apu-8U.

A 1.1-kb sequence containing an αAmy3 promoter-signal peptide sequencewas excised with SalI and HindIII from p3G-13211 (Lu et al., 1998, J.Biol. Chem. 273, 10120-10131.) and subcloned into pBluescript togenerate pBS-3sp. The αAmy3 3′UTR was excised with HindIII and SacI frompMTC37 (Chan and Yu, 1998, Plant J. 15:685-696.) and subcloned into thecorresponding sites in pBS-3sp to generate pBS-3sp3U. Theabove-described truncated apu sequence was digested with BamHI and NotIand subcloned into the same sites in pBS-3sp3U to generatepBS-αAmy3-sp-Apu-3U.

DNA sequencing was conducted and confirmed that the above-describedligations were correct or in-frame. Then, the GluB-sp-Apu-Nos,αAmy3-sp-Apu-αAmy3 3′UTR, and αAmy8-sp-Apu-αAmy8 3′UTR chimeric geneswere excised from pBS-Gp-Apu-Nos, pBS-αAmy3-sp-Apu-3U, andpBS-αAmy8-sp-Apu-8U with SalI, blunt-ended, and inserted into theHindIII-digested and blunt-ended binary vector pSMY1H (Ho et al., 2000,Plant Physiol. 122, 57-66) to generate pGpApu, pA3Apu and pA8Apu,respectively.

Transgenic rice

The above-described plasmids pGpApu, pA3Apu, and pA8Apu, wererespectively introduced into Agrobacterium tumefaciens strain EHA101(Hood et al., 1986, J. Bacteriol. 168,1291-1301) with an electroporator(BTX) according to the manufacturer's instruction and delivered to therice genome. Calli induced from immature rice seeds were transformedwith Agrobacterium according to the methods described by Hiei et al.,1994, Plant J. 6:271-282; and Toki, 1997, Plant Mol. Biol. Rep. 15,16-21. Transformed rice calli were then selected on a medium containinghygromycin.

Identity of the transformed rice cells was confirmed with genomic DNASouthern blot analysis. More specifically, rice seeds were germinatedand grown in the dark for 1 week. Genomic DNA was isolated from the wildtype or transformed calli according to the method described in Sheu etal., 1996, J Biol. Chem. 271:26998-27004. Ten μg of genomic DNA wasdigested with restriction enzymes, fractionated in 0.8% agarose gel, andtransferred to a nylon membrane (MSI). Hybridization was performed at42° C. using a ³²P random primer labeled APU cDNA probe.

Expression of APU in E. coli and preparation of polyclonal antibodies

APU was expressed in E. coli. More specifically, the sequence encodingApu amino acids 75 to 1029 was PCR-amplified from genomic DNA of T.ethanolicus 39E (ATCC53033), which was prepared according to the methodof Sheu et al. just mentioned. Primers used were5′-CGCATATGTTAAGCTTGCATCTTG-ATTC-3′ (forward primer, NdeI siteunderlined) and 5′-CCGCTCGAGCTACATATTTTC-CCCTTGGCCA-3′ (reverse primer,XhoI site underlined). The amplified DNA fragment was digested with NdeIand XhoI and ligated into the same sites in pET20b(+) (Novagen) togenerate pET-APU. After introducing pET-APU into E. coli strain BL21(DE3), APU protein was expressed and purified according to theinstruction provided by Novagen. The E. coli expressed APU has extra 6histidines and 25 amino acid residues at C terminal, which increase themolecular weight of APU by about 3.4 kD. One hundred μg of purified APUwere injected into a New Zealand White rabbit at 4-6 week interval togenerate polyclonal antibodies according to the methods described inWilliams et al., 1995, In: DNA Cloning 2. Expression Systems. APractical approach. (Ed) Glover D M and Hames B D. IRL Press, NewYork.).

APU expression in transformed rice suspension cells is sugar-regulated

The above-described transformed rice calli were cultured in a liquid MSmedium to generate a suspension cell culture. Culture media of cellsexpressing APU fused with signal peptides were collected and analyzed byboth Enzyme-linked immunosorbent assay (ELISA) and protein gel blotanalyses according to the methods described in Ausubel et al. 1992,Short Protocols in Molecular Biology. 2^(nd) edit. In: A Compendium ofMethods from Current Protocols in Molecular Biology. John Wiley & Sons.New York.).

In the ELISA, the E. coli-expressed APU was used as a standard. Thepercentage APU in total medial proteins was then obtained. The totalprotein concentration was determined using a Bio-Rad protein assay kitbased on the dye-binding assay of Bradford (Bradford, 1976, Anal Biochem72:248-254.). In the protein gel blot analysis, total proteins wereextracted from cultured suspension cells with an extraction buffer (50mM Tris-HCl, pH 8.8, 1 mM EDTA, 10% glycerol, 1% Triton X-100, 10 mMβ-mercaptoethanol, and 0.1% sarkosyl). The culture medium was collectedand centrifuged at 18,000 g at 4° C. for 15 min to remove cell debris.Western blot analysis was performed as described in Yu et al., 1991, J.Biol. Chem. 266:21131-21137.

The results show that relatively high level expressions of APU werefound in media of transformed suspension cells and no APU was detectedin media of non-transformed cells. The levels of APU varied from line toline, possibly due to a position effect or multiple copy gene effect ontransgene expression. The presence of APU in the culture media indicatesthat the putative signal sequence of GluB-1 is capable of directingtranslocation of APU through the secretary pathway. The αAmy3 and αAmy8promoters directed higher levels of APU expression in the absence ofsucrose than in the presence of sucrose. This result was expected asactivity of αAmy3 and αAmy8 promoters is up-regulated by sucrosestarvation (Lu et al., 1998, J. Biol. Chem. 273, 10120-10131).Interestingly, the GluB-1 promoter directed a higher level of APUexpression in rice suspension cells in the presence of sucrose than inthe absence of sucrose, suggesting that the activity of the GluB-1promoter is up regulated by sucrose in cultured rice suspension cells.

The molecular weight of APU expressed by the transformed rice cells issimilar as that expressed by E. Coli, indicating that the APU expressedby rice cells are approximately 3.4 kD larger than the theoreticmolecular weight. Since APU has three potential glycosylation sites,this result suggests that glycosylation of APU has occurred in ricecells.

Further analysis indicated that high expression levels APU were obtainedunder the control of an a-amylase promoter (in the absence of sucrose)and the above-described glutelin promoter (in the presence of sucrose).The highest levels were up to 12% of total proteins, which are muchhigher than those of the E.coli repression system described above. Thisresult suggests that these two promoters have similar efficiency indirecting expression of recombinant proteins in cultured suspensioncells.

EXAMPLE 2

GluB-1 promoters containing SEQ ID NO: 9 or 14 were used to generatetransformed rice cells and transgenic rice. The rice variety used inthis example was the s Oryza sativa L. cv. Tainung 67. Immature seedswere dehulled, sterilized with 2.4% NaOCl for 1 hour, washed extensivelywith sterile water, and placed on CIM agar medium (Toki, 1997, PlantMol. Biol. Rep. 15, 16-21) for callus induction. After one month, calliderived from scutella were subcultured in a fresh CIM medium fortransformation.

Plasmid construction

The 1351-bp rice GluB-1 promoter described above in Example 1 wasPCR-amplified and subcloned into pBluescript to generate pBS/GluB-1,which was subsequently used as a template for all PCR-based mutagenesis.Modification of the GluB-1 promoter was carried out by a two-step PCRsequence substitution method, using P1 and P2 as primers in the primaryPCR for generating a megaprimer P3 and then using P3 and P4 primers inthe secondary PCR for generating modified promoter regions. The P2(5′-CGCGATATCGTACTTGCTTATGG-3′, EcoRV site underlined) and P4(5′-GTCATGGCTGAGTCATGAAATG-3′, BspHI site underlined) primers were usedin PCR for each modified GluB-1 promoter.

For construction of a GM1 promoter, in which the TA box-like (TATCCC)sequence in the GluB-1 promoter was substituted by two tandemly repeatedTA boxes, P1 (GM 1) primer (5 ′-ATATATTGTCTGTACGTGCAACAAACTATATCACCATGTATCCATATCCAAAGATGCTTTTTTATTGCTAT-3′, tandemly repeated TA boxunderlined) and P2 primer were used for amplification of fragment M1.For construction of a GM2 promoter, in which sequences flanking thetandemly repeated TA box in the GM1 promoter were substituted withsequences flanking TA box in SRS, P1 (GM2) primer(5′-ATATATTGTCTGTACGTGCAACAAACTATATCACCcctTATCCATATCCACgccTTTATTGCTAT-3′, TA box underlined and modified flankingsequences in bold lowercase) and P2 primer were used for amplificationof fragment M2. For construction of a GM3 promoter, in which sequencesflanking the G-box in the GM2 promoter were substituted with sequencesflanking the G box in SRS, P1 (GM3) primer(5′-ATATATTGTCccTACGTGgcACAAACTATATCACCcctTATCCATATCCACgccTTTATTGCTAT-3′, G box and TA box underlined and modified flankingsequences in bold lowercase) and P2 primer were used for amplificationof fragment M3. DNA fragment between the BspHI and EcoRV sites in theGluB-1 promoter was substituted by fragment M1, M2, or M3 to generatepBS/GM1, pBS/GM2, and pBS/GM3, respectively.

To fuse the luciferase (Luc) gene downstream of the wild type ormodified GluB-1 promoter, a Luc-Nos 3′ fragment was isolated from pJD312(Luehrsen et al., Methods Enzymol. 216:397-414) with SalI and Bgl II andinserted into the SalI and BamHI sites of pBS to generate pBS/LN. TheLuc-Nos3′ fragment was then excised from pBS/LN by SalI and XbaI,end-blunted, and inserted into the EcoRV site in pBS/GluB-1, pBS/GM 1,pBS/GM2, and pBS/GM3 to generate pBS/GLN, pBS/GM1LN, pBS/GM2LN, andpBS/GM3LN, respectively.

A sequence was excised with EcoRI from pTRA151 (Zheng et al., 1991,Plant Physiol. 97:832-835). This sequence contained a cauliflower mosaicvirus 35S RNA (CaMV35S) promoter-hygromycin B phosphotransferase (Hpt)coding sequence-tumor morphology large gene 3′UTR (tml) fusion gene. Itwas then subcloned into the EcoRI site of the binary vactor pPZP200(Hajdukiewicz et al., 1994, Plant Mol. Biol. 25: 989-994) to generatepPZP/HPH. pPZP/HPH was then linearized with KpnI and served as a vectorbackbone. pBS/GLN, pBS/GM1LN, pBS/GM2LN, and pBS/GM3LN were linearizedwith KpnI and respectively inserted into this vector backbone togenerate pGLN, pGM1LN, pGM2LN, and pGM3LN, respectively.

Transgenic rice

The just-described pGLN, pGM1LN, pGM2LN, and pGM3LN were introduced intorice using Agrobacterium tumefaciens strain EHA101 in the same mannerdescribed above in Example 1. Each of the resultant transgenic lines wasthen examined for the copy number of transgene in its genome by DNA gelblot analysis. More specifically, genomic DNA was isolated from riceleaves according to the method described in Sheu et al., 1996, J. Biol.Chem. 271:26998-27004. Ten μg genomic DNA was digested with SpeI,separated in a 0.7% agarose gel, and transferred to a nylon membrane(MSI). The membrane was then hybridized with a 2 kb ³²P-labeled Luc-Nos3 ′DNA fragment probe. It was found that each of the transgenic lineshad about 1-5 copies of the transgene, except that GLN-2 seemed to havemore than 5 copies.

Suspension cell culture and promoter analysis

Calli were prepared from the just-described transgenic rice plants usingstandard techniques and were selected for bhygromycin resistance toestablish suspension cells. The cells were cultured on a reciprocalshaker at 120 rpm and incubated at 26° C. in dark. They were subculturedevery 7 days by transferring 0.5 mL of the cell culture into 25 mL offresh liquid MS medium in a 125-mL flask. To examine the regulationeffects of sugar, sucrose was added to the callus culture (in a CIM ) orsuspension cell culture (in an MS liquid medium) to reach a finalconcentration of 3%. After an incubation of 24 hours at 28° C. in thedark, 0.5 g callus or suspension cell culture were collected forpromoter analysis.

More specifically, total proteins were extracted from calli, culturedsuspension cells, or matured rice grains using a CCLR buffer (100 mMKH₂PO₄, pH 7.8, 1 mM EDTA, 10% glycerol, 1% Triton X- 100, 7 mMβ-mercaptoethanol). Protein concentrations were determined using aCoomassie protein assay reagent (Bio-Rad). The activity of each GluB-1promoter was examined using luciferase activity assay according to themethod described in Lu et al., 1998, J. Biol. Chem. 273:10120-10131.

It was found that, in all three types of cells, the above-describedthree modified promoters (GM1, GM2, and GM3) exhibited stronger promoteractivity than the promoter having the above-described 1351 bp region(GluB-1) in either absence or presence of sucrose. In general, promoteractivities were higher in the absence of sucrose than in the presence ofsucrose. Based on their promoter activities, the promoters were rankedfrom the strongest to the weakest. The ranking is shown in Table 1below: TABLE 1 Promoter strength ranking Cell Type Promoter StrengthRice calli GM3 > GM2 > GM1 > GluB-1 Rice suspension cells in presence ofsucrose GM3 > GM2 > GM1 > GluB-1 in absence of sucrose GM3 > GM1 > GM2 >GluB-1 Rice seeds GM3 > GM2 > GM1 > GluB-1

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the scope of thefollowing claims.

1. A nucleic acid comprising a GluB-1 promoter that contains thesequence of SEQ ID NO: 9 or 14, or the complement thereof
 2. The nucleicacid of claim 1, wherein the GluB-1 promoter contains SEQ ID NO: 10, orthe complement thereof.
 3. The nucleic acid of claim 2, wherein theGluB-1 promoter contains SEQ ID NO: 4, or the complement thereof.
 4. Thenucleic acid of claim 3, wherein the GluB-1 promoter contains SEQ ID NO:15 or 18, or the complement thereof.
 5. The nucleic acid of claim 1,wherein the GluB-1 promoter contains SEQ ID NO: 11, or the complementthereof.
 6. The nucleic acid of claim 5, wherein the GluB-1 promoter SEQID NO: 5, or the complement thereof.
 7. The nucleic acid of claim 6,wherein the GluB-1 promoter contains SEQ ID NO: 16 or 19, or thecomplement thereof.
 8. The nucleic acid of claim 1, wherein the GluB-1promoter contains SEQ ID NOs: 9 and 14, or the complement thereof. 9.The nucleic acid of claim 8, wherein the GluB-1 promoter contains SEQ IDNO: 6, or the complement thereof.
 10. The nucleic acid of claim 9,wherein the GluB-1 promoter contains SEQ ID NO: 17 or 20, or thecomplement thereof.
 11. A vector comprising the nucleic acid of claim 1.12. A transformed plant cell comprising a promoter sequence thatcontains the nucleic acid of claim 1; and a recombinant nucleic acidthat encodes a heterologous protein, wherein the promoter sequence isoperatively linked to the recombinant nucleic acid.
 13. The plant cellof claim 12, wherein the plant cell is a monocot plant cell.
 14. Theplant cell of claim 13, wherein the plant cell is a cereal plant cell.15. The plant cell of claim 14, wherein the plant cell is a rice cell, acorn cell, a wheat cell, a barley cell, an oat cell, or a sorghum cell.16. The plant cell of claim 12, wherein the plant cell is a dicot plantcell.
 17. The plant cell of claim 16, wherein the plant cell is atobacco cell, a potato cell, a tomato cell, or a soybean cell. 18 Atransgenic plant comprising a genome that includes a promoter sequencethat contains the nucleic acid of claim 1; and a recombinant nucleicacid that encodes a heterologous protein, wherein the promoter sequenceis operatively linked to the recombinant nucleic acid.
 19. Thetransgenic plant of claim 18, wherein the plant is a monocot plant. 20.The transgenic plant of claim 19, wherein the plant is a cereal plant.21. The transgenic plant of claim 20, wherein the plant is rice, corn,wheat, barley, oat, or sorghum.
 22. The transgenic plant of claim 18,wherein the plant is a dicot plant.
 23. The transgenic plant of claim22, wherein the plant is tobacco, potato, tomato, or soybean.
 24. Amethod of producing a transformed plant cell, the method comprisingintroducing into a plant cell a promoter sequence that contains thenucleic acid of claim 1; and a recombinant nucleic acid that encodes aheterologous protein, wherein the promoter sequence is operativelylinked to the recombinant nucleic acid.
 25. A method of producing atransgenic plant, the method comprising: introducing into a plant cell apromoter sequence that contains the nucleic acid of claim 1; and arecombinant nucleic acid that encodes a heterologous protein, whereinthe promoter sequence is operatively linked to the recombinant nucleicacid; and cultivating the cell to generate a plant.
 26. An isolatednucleic acid comprising the sequence of SEQ ID NO: 1 or the complementthereof, wherein the nucleic acid is 1,307 to 2,300 nucleotides inlength
 27. A vector comprising the nucleic acid of claim
 26. 28. Atransformed plant cell comprising: a promoter sequence that contains thenucleic acid of claim 26; and a recombinant nucleic acid that encodes aheterologous protein, wherein the promoter sequence is operativelylinked to the recombinant nucleic acid.
 29. The plant cell of claim 28,wherein the plant cell is a monocot plant cell.
 30. The plant cell ofclaim 29, wherein the plant cell is a cereal plant cell.
 31. The plantcell of claim 30, wherein the plant cell is a rice cell, a corn cell, awheat cell, a barley cell, an oat cell, or a sorghum cell.
 32. The plantcell of claim 28, wherein the plant cell is a dicot plant cell.
 33. Theplant cell of claim 32, wherein the plant cell is a tobacco cell, apotato cell, a tomato cell, or a soybean cell.
 34. The plant cell ofclaim 28, wherein the promoter sequence contains SEQ ID NO:
 2. 35. Atransgenic plant comprising genome that includes a promoter sequencethat contains the nucleic acid of claim 26; and a recombinant nucleicacid that encodes a heterologous protein, wherein the promoter sequenceis operatively linked to the recombinant nucleic acid.
 36. Thetransgenic plant of claim 35, wherein the plant is a monocot plant. 37.The transgenic plant of claim 36, wherein the plant is a cereal plant.38. The transgenic plant of claim 37, wherein the plant is rice, corn,wheat cell, barley, oat, or sorghum.
 39. The transgenic plant of claim35, wherein the plant is a dicot plant.
 40. The transgenic plant ofclaim 39, wherein the plant is tobacco, potato, tomato, or soybean. 41.The plant of claim 35, wherein the promoter sequence contains SEQ ID NO:2.
 42. A method of producing a transformed plant cell, the methodcomprising: introducing into a plant cell a promoter sequence thatcontains the nucleic acid of claim 26, and a recombinant nucleic acidthat encodes a heterologous protein; and expressing the heterologousprotein in the cell, wherein the promoter sequence is operatively linkedto the recombinant nucleic acid.
 43. A method of producing a transgenicplant, the method comprising: introducing into a plant cell a promotersequence that contains the nucleic acid of claim 26, and a recombinantnucleic acid that encodes a heterologous protein; and cultivating thecell to generate a plant, wherein the promoter sequence is operativelylinked to the recombinant nucleic acid.
 44. A method of expressing apolypeptide in a cell, the method comprising introducing into a hostcell a recombinant nucleic acid encoding a polypeptide, wherein therecombinant nucleic acid is operatively linked to a GluB-1 promotersequence; and culturing the host cell under conditions permittingexpression of the polypeptide.
 45. The method of claim 44, wherein thehost cell is cultured in presence or absence of sugar.
 46. The method ofclaim 44, further comprising recovering the polypeptide.